EP1711204A1

EP1711204A1 - Monoparaimmunity inducers based on attenuated rabbit myxoma viruses

Info

Publication number: EP1711204A1
Application number: EP05701103A
Authority: EP
Inventors: Anton Mayr; Barbara Mayr
Original assignee: Bavarian Nordic AS
Current assignee: Bavarian Nordic AS
Priority date: 2004-01-23
Filing date: 2005-01-21
Publication date: 2006-10-18
Also published as: US7939085B2; IL176634A0; EP2327420A2; CA2547757A1; WO2005070453A8; US20070154455A1; AU2005205912B2; US20120070464A1; KR20120076396A; US20080213307A1; US7494799B2; HK1095746A1; US20080267920A1; CN1909923B; US7691391B2; US20110014234A1; IL176634A; WO2005070453A1; KR20060129221A; US8039004B2

Abstract

The invention relates to monoparamunity inducers that are based on paramunizing viruses or viral components of a myxoma virus strain of diseased rabbits that show the general symptoms of the disease. The invention also relates to a method for producing the inventive inducers and to their use as drugs for the regulatory optimization of the paramunizing activities for use in the prophylaxis and therapy of various human and animal dysfunctions.

Description

MONOPARAMUNITY INDUCERS BASED ON ATTENUATED MYXOMAVIRES OF THE RABBIT

The invention relates to monoparamunity inducers based on paramunizing viruses or virus components, characterized in that the viruses or virus components originate from an attenuated myxomavirus strain of the rabbit, a method for producing the monoparamity inducers and their use as medicaments.

The body's immune system of highly developed organisms, especially that of mammals and birds, includes an antigen-specific and an antigen-unspecific part. Both parts of the immune system are networked and interact with each other. The antigen-specific mechanisms are responsible for the build-up of immunity, the antigen-specific ones for the build-up of paramunity. Paramunity refers to the state of a well-regulated and optimally functioning non-specific defense system, combined with a rapidly emerging, time-limited, increased protection against a large number of different pathogens, antigens and other noxious agents. For historical and functional reasons, the basis for the emergence of paramunity is the so-called primitive, non-selective and conditionally selective, parasitic defense mechanisms that are old from a phylogenetic point of view.

The paraspecific activities of the antigen-unspecific immune system (English: "innate immune system") include non-selective protective elements, such as foreign material-eating organelles, and limited selective protective elements, such as micro- and macrophages, natural killer cells, dendritic cells and soluble factors such as cytokines, which react pathogen-unspecific or antigen-unspecific depending on their genesis.

Paraspecific activities can be observed in the organism concerned immediately after antigen contact, while the effects of the antigen-specific immune system only take days or weeks.

In the case of the more highly organized organisms, additional time is gained in this way to build up specific defense systems against the noxae, which have not yet been eliminated and which have antigenic properties. The benefits of paramunization, ie the paraspecific activities of the immune system for prophylaxis and therapy in a patient, have been shown ever more clearly since their development (Anton Mayr, "Paramunization: empiricism or science", Biol. Med., Edition 26 ( 6): 256-261, 1997) The parasitic defense enables the organism to immediately defend itself when confronted with a wide variety of foreign substances, infectious agents, toxins and transformed body cells.

There are close interactions between the paraspecific and the specific activities of the immune system, whereby the information flow usually runs from the first reacting, paraspecific to the later onset, specific part of the immune system (e.g. in antigen mediation). In this way, the parasitic defense of the organism in the case of infections with particularly virulent pathogens can bridge the time until the development of specific immunity (e.g. antibodies, immune cells).

The paraspecific immune defense is a physiological process and can be defined as "primary control" when dealing with the environment. It is irreplaceable not only for the lower organisms, but especially for the more highly and highly developed vertebrates. Primary congenital defects in this biological Defense systems lead to life-threatening situations. One example is the "Chediak-Steinbrinck-Higashi syndrome" of humans, which is characterized by granulocyte defects and dysfunctions of the natural killer cells (NK cells) and in most cases until the 10th Leads to the death of the patient.

The state of paramunity is characterized by an increased phagocytosis rate, an increased function of spontaneous cell-mediated cytotoxicity (NK cells) and an increased activity of other lymphoreticular cells. At the same time, certain cytokines are released, which have a stimulating and / or inhibiting effect (for example via repressor mechanisms) both with the cellular elements and with one another. This closely networked and gradually reacting biological system of paramunity with its different acceptor, effector and target cells as well as the signal-transmitting cytokines is also closely connected to the hormone and nervous system. It is an important part of the communication, interaction and regulation network. Paramunity is initiated by paramunization. This means the medicinal activation of the cellular elements of the paraspecific part of the immune system and the associated formation of cytokines, with the aim of eliminating dysfunctions, quickly increasing the non-pathogen and non-antigen-specific protection of an individual (optimal bioregulation), one due to stress consequences or to eliminate immunosuppression or immune deficiency that has arisen in some other way (for example with medication), to repair deficits and / or to act in a regulatory manner between the immune, hormone and nervous systems. This means that, depending on the type of parameterization and the reaction situation, such as the patient's health situation, certain non-specific, body-specific defense processes can be increased, supplemented or even dampened.

Paramunization inducers are used for paramunization, to which certain harmlessness and efficacy criteria are set, which distinguish them from immunostimulants. The paramunity inducer per se is not comparable to a repellent, nor to a chemical, an antibiotic, vitamin or hormone. Rather, it activates the paraspecific immune system via a step-by-step mechanism, so that it mobilizes sufficient cellular and humoral defense mechanisms. The paramunity inducer acts both regulating and repairing with regard to the immune defense. With regard to the mode of action of paramunity inducers, it is known that they are taken up by phagocytic cells (acceptor cells), which are thereby activated and release mediators, which in turn mobilize effector cells. These finally switch on the regulatory mechanisms of the parasitic defense.

Multiple paramunity inducers based on combinations of two or more smallpox virus components, which are derived from different poxvirus strains with paramunizing properties, are described in European Patent EP 0 669 133 B1.

The present invention is based on the paramunizing properties of attenuated myxoma viruses and / or their virus components.

Attenuation refers to the transformation of the properties of an infectious agent that lead to the weakening of the infectious agent (English: "attenuate" = weaken, mitigate). Changes in infectious agents occur spontaneously in nature frequently, and the time span can extend over many centuries. Experimentally, the adaptability of infectious agents to adapt to environmental changes can be used and the time required for attenuation can be drastically shortened, for example through permanent passages in certain host systems. So far, attenuation has been used to obtain avirulent vaccine strains and harmless paramunity inducers.

As a rule, attenuation leads to loss of virulence and contagiosity, a reduction in the immunizing properties and the host spectrum and to minor changes in the pathogen genome with the appearance of deletions, preferably in the terminal areas. At the same time, there is usually an increase in the paramunizing activities of the modified pathogen.

In rare cases, attenuation, especially when attempting experimental attenuation through genetic engineering manipulations, can also lead to an increase in virulence and contagiosity.

Myxoma viruses are the causative agents of myxomatosis, a cyclical, contagious viral general disease of wild and domestic rabbits, which is characterized by generalized, partially hemorrhagic subcutaneous edema on the head and all over the body, with preference for the anal region, the vulva and the tube, like no other infectious disease is. If myxomatosis is newly introduced into a previously disease-free country, it is rapid and fatal. After the virus becomes sedentary, the epidemic character changes up to clinically unapparent infections (Mayr A .: Medical microbiology, infection and epidemics, 7th edition, Enke-Verlag, Stuttgart, 2002).

The disease is widespread among American cottontail rabbits of the genus Sylvilagus, which only colonize the new world. These wild rabbits are the only natural reservoir of the disease. The infection is mild. In contrast, the disease in European wild and domestic rabbits of the genus Oryctolagus, which are also native to Australia, has an almost 100% mortality rate when the pathogen is newly introduced.

The natural host spectrum of the Myxomavirus (genus Leporipoxvirus) is narrowly limited. In general, the virus only reproduces in American cottontail rabbits and in European domestic and wild rabbits. However, were also isolated Infections in European wild rabbits observed. Attempts to transfer it to other animal species and to humans were negative.

The present invention has for its object to provide new monoparamunity inducers for human medicine and veterinary medicine. Another object of the present invention is to provide a method for producing such monoparamity inducers. It is also an object of the present invention to provide pharmaceutical compositions for use as medicaments based on monoparamity inducers.

Accordingly, the present invention relates to monoparamunity inducers based on paramunizing viruses or virus components, characterized in that the viruses or virus components originate from an attenuated myxoma virus strain of the rabbit. The virus components preferably comprise paramunizing virus envelopes or aberrant forms of virus envelopes of an attenuated myxoma virus strain. Preferred strains which have the parameterizing properties according to the invention are the strains M-2, M-7, Lausanne, Aust / Uriarra / Verg-86 /. The strains M-7, Lausanne, Aust / Uriarra / Verg-86/1 are also suitable for the production of live vaccines, since their virulence is only partially weakened in order to have a sufficiently immunizing effect.

A monoparamity inducer based on the myxomavirus strain M-2 is particularly preferred. An attenuated myxoma virus strain, produced by the method according to the invention described below, is available at the depository of the Public Health Laboratory Service (PHLS), Center for Applied Microbiology & Research, European Collection of Animal Cell Cultures (ECACC), Salisbury, Wiltshire, United Kingdom the deposit number 03121801.

The invention further relates to a method for producing monoparamunity inducers based on an attenuated myxomavirus strain of the rabbit. To do this, myxoma viruses are first isolated from infected tissue of a rabbit typically suffering from generalized myxomatosis. The virus is then adapted to a permissive cell system, ie to a cell material that allows the virus to multiply, such as cell cultures, incubated chicken eggs or experimental animals. In particular, cells from the natural host or a species closely related to the host can be used for adaptation. As permissive cell systems for myxoma viruses For example, chicken embryo fibroblasts (FHE) are suitable, as are cell cultures made from rabbit kidneys or testicles.

Preferred according to the invention is the adaptation of the isolated myxoma virus to the chorioallantoic membrane (CAM) of incubated chicken eggs over one or more passages, preferably over 2 to 6 passages and particularly preferably over 3 passages. For this purpose, the isolated viruses are vaccinated on the CAM and multiplied by passing through the CAM.

The myxoma viruses are preferably first isolated from infected tissue by multiplication in a permissive cell system. For this purpose, a permissive cell system can be inoculated, for example, with infected tissue homogenate obtained by digestion. Subsequently, the viruses obtained by first propagation are either further adapted to the same permissive cell system type that was already used for the isolation, or a different, further permissive cell system is used. The adaptation of the virus to the same permissive cell system type that is also used for the isolation is preferred. Myxoma viruses are thus preferably isolated from infected tissue by multiplication in a permissive cell system and then adapted to the permissive cell system by further passages. Particularly preferred is a first cultivation or isolation of the myxoma viruses by multiplying chicken eggs incubated on the CAM and subsequent adaptation of the virus on the CAM over further passages, preferably over a further 2 passages. Alternatively, the allantoic liquid of incubated chicken eggs can also be used for the cultivation and / or adaptation.

The actual attenuation then takes place through permanent passages on one or more permissive cell cultures until attenuation or the desired degree of attenuation of the virus is achieved. To do this, various permissive cell systems can first be tried out to multiply the virus, and then one or more cell systems in which the highest infectivity titers are achieved can be selected for further passages. Both primary and secondary cell cultures as well as permanent or continuous cell lines are suitable for attenuation through long-term passages. For example, attenuation can be achieved by multiplication in primary or secondary chicken embryo fibroblast (FHE) cultures or in cultures of permanent FHE cells.

According to the invention, the virus is preferably passaged or propagated in a permanent cell culture, in particular a Vero cell culture, for attenuation of the myxoma virus, preferably over 80 to 150 passages and particularly preferably over 120 passages. Alternatively or additionally, the virus is passaged according to the invention on a binary, permanent cell line, AVIVER cells preferably being used. This cell or cell culture was obtained by a cell fusion between chicken embry fibroblasts (FHE) and Vero monkey kidney cells. For the attenuation of the myxoma virus according to the invention, the isolated and adapted viruses are preferably passaged in a first step in Vero cell cultures, the viruses are subsequently transferred into a binary AVIVER cell culture and in these preferably over 10 to 50 passages, in particular over 20 to 30 passages, and particularly preferably increased over 25 passages.

Subsequently, the attenuated myxoma virus can be multiplied by additional attenuation passages. A further multiplication of the viruses takes place preferably via further passage in Vero monkey kidney cells, in particular over 100 to 200 passages.

A particularly preferred, further process step is an additional inactivation of the attenuated myxoma virus. Inactivation can take place by chemical treatment, radiation, exposure to heat or pH, in particular by chemical treatment with beta-propiolactone. By treating the attenuated myxoma viruses with beta-propiolactone, the paraspecific activities are increased, while the immunizing properties that may still be present after attenuation are lost.

A particularly preferred embodiment of the method according to the invention comprises the following steps: isolating myxoma viruses from infected tissue of a rabbit typically suffering from generalized myxomatosis by multiplying chicken eggs incubated on the chorioallantoic membrane (CAM) and subsequently adapting the virus to the CAM over a further 2 passages; - attenuation of the isolated viruses by passage in Vero cell cultures, preferably over 120 passages; Transfer of the viruses into a binary AVIVER cell culture, the AVIVER cells being obtained by a cell fusion between chicken embryo fibroblasts (FHE) and Vero monkey kidney cells, and attenuation of the virus in this cell culture over 10 to 50, preferably 25, passages. subsequent retransfer of the virus to Vero monkey kidney cells and multiplication of the viruses by further attenuation passages in the Vero cells, preferably over approximately 150 passages;

and optionally

Inactivate the attenuated myxoma viruses by treatment with beta-propiolactone, this treatment increasing the paraspecific activities, while the immunizing properties still present after attenuation are lost.

Attenuation through permanent passage is usually completed by 3 to 5 final plaque dilutions. After obtaining and testing different clones, those clones are selected for further propagation with which the highest infectivity titer is achieved and with which a high paramunizing effectiveness - e.g. in the VSV challenge test in the baby mouse. This procedure is intended in particular to provide genetically uniform virus material for further use.

The term "attenuation", as used in connection with this invention, denotes the experimental change of the originally virulent myxoma virus into the modified form, while simultaneously increasing the paramunizing properties. Attenuation can be demonstrated by one or more of the following properties: reduction or Weakening or loss of virulence for European domestic and wild rabbits (genus Oryctolagus caniculus csp.), Weakening or loss of contagiosity, narrowing of the host spectrum in cell cultures, change in the immunizing properties, acquisition of paramunizing, short-term protective activities. Attenuation can lead to deletions in the terminal Area of the myxoma virus genome, and with increasing degree of attenuation an increasing number of deletions in the viral genome is observed.

The degree of attenuation can be checked and checked in the course of the passages by appropriate, suitable effectiveness tests, as are known in the prior art (cf., for example, US Pat. No. 6,805,870, column 12, and the further literature references cited therein) and by cloning. The present invention further relates to attenuated myxomaviruses which can be obtained by the process according to the invention, pharmaceutical compositions which comprise the attenuated myxomavirus or the myxomavirus monoparamunity inducer according to the invention, and the use of the myxomavirus monoparamunity inducer for activating the paraspecific immune system in a mammal or Use of the attenuated virus for the manufacture of a corresponding drug.

Because of the surprising paramunizing properties, the myxomavirus monoparamunity inducers according to the invention are suitable for the treatment and / or prophylaxis of immune system dysfunctions, immunosuppression, immune deficiency disorders, dysfunctions of homeostasis between hormone, circulatory, metabolic and nervous systems, neonatal infection threats, tumor diseases, Viral diseases, bacterial diseases, therapy-resistant infectious factor diseases, viral and bacterial mixed infections, chronic manifestations of infectious processes, liver diseases of different origins, chronic skin diseases, herpes diseases, chronic hepatitis, flu infections, endotoxin damage.

The monoparamity inducers according to the invention are generally harmless to the environment and are effective in terms of paramunization for mammals, e.g. Human, horse, dog, cat, pig, for birds and also for reptiles, such as Lizards, snakes, turtles. They are therefore particularly well suited for use in human and veterinary medicine.

In addition, the monoparamunity inducers according to the invention have very good paramunizing activity with high potency. They can be produced in a suitable manner using the method according to the invention and are safe for use in the medical field. As a result of the attenuation of the myxoma viruses, the immunizing properties of the myxoma viruses decrease, while the paraspecific activities increase. The monoparamity inducers according to the invention therefore have no immunizing, but rather paramunizing properties, which enables repeated and continuous use. These paramunizing properties of the rabbit myxoma virus or its paramunizing virus components are surprising and were not predictable. The term "paramunization" as used in connection with this invention denotes the medicinal activation of the cellular elements of the paraspecific immune system and the associated formation or release of cytokines with the aim of eliminating dysfunctions, the non-pathogen and non-antigen-specific protection of To increase the individual quickly and to act as a regulator between the immune, hormone, nervous and vascular systems: Paramunization leads to the protective state of the paramunity.

The term “paramunity” as used in connection with this invention denotes the actively acquired state of an optimally regulated and functioning paraspecific defense system, combined with a rapidly emerging, time-limited protection against a large number of pathogens, antigens and other noxious substances. The phagocytosis rate, the function of the NK cells (natural killer cells) and the activity of other lymphoreticular cells (e.g. dendritic cells) are increased to the physiological optimum.

The term "paramunity inducer" as used in connection with this invention denotes a pyrogen-free, non-toxic medicament which is intended to be used in humans and animals for the production and regulation of the body's own defense and protection mechanisms in the sense of a paramunization.

The term "myxomavirus monoparamity inducer" as used in connection with this invention refers to a medicament based on rabbit attenuated myxoma viruses or an attenuated myxoma virus strain, including the paramunizing virus components and the components thereof, which are preferred in an organism in a mammal (e.g. human) to produce the state of paramunity.

The term "myxomavirus" as used in connection with this invention denotes the species of the myxomatosis virus of the genus Leponpoxvirus. The myxomavirus belongs to the subfamily of the Chordopoxviridae and the family of the Poxvi dae (smallpox viruses).

The term "paramunizing virus components" as used in the context of this invention encompasses a large number of viral structures which are derived from a myxoma virus with paramunizing properties, for example reproductive or inactivated freshly isolated myxoma viruses, reproductive or inactivated recombinant myxoma viruses, which derived from freshly isolated myxoma viruses, Virus envelopes, the separated envelopes, and cleavage products and aberrant forms of these envelopes, individual native or recombinant polypeptides or proteins, in particular membrane and surface receptors, which occur in freshly isolated myxoma viruses or are recombinantly expressed by a genetically modified myxoma virus or part of its genetic information.

Tables 1 to 4 summarize the clinical results with the myxoma virus monoparamunity inducer PIND-MYXO based on the attenuated myxomatosis cell culture virus, strain M-2, in humans.

Table 1 shows the clinical results in the prophylactic use of the myxomavirus monoparamity inducer PIND-MYXO in humans.

Table 2 shows the clinical results in the therapeutic use of the myxoma virus monoparamunity inducer PIND-MYXO in humans.

Table 3 shows the effect of paramunization with myxomavirus monoparamunity inducer (PIND-MYXO) in patients with low immune parameters (7 days after PIND-MYXO application)

Table 4 shows the effect of paramunization with myxomavirus monoparamunity inducer (PIND-MYXO) in patients with increased immune parameters (7 days after PIND-MYXO application)

The invention is based on the surprising finding that attenuated rabbit myxoma viruses or their paramunizing components are able to produce very good paramunizing properties in a recipient organism, which lead to the protective state of the paramunity. The basis for this invention was the first successful attenuation of rabbit myxoma viruses in cell cultures.

The monoparamity inducers according to the invention are preferably based on lyophilized, attenuated and inactivated myxoma viruses of the rabbit or their paramunizing virus components. The attenuated myxomaviruses according to the invention or their virus components preferably originate from one myxomavirus strain or from several different attenuated myxomavirus strains. It is preferred that the monoparamity inducer according to the invention combinations of one or more strains of myxoma virus or their paramunizing virus components.

The paramunizing properties caused by the application of myxomavirus monoparamunity inducer in a mammal, such as in humans, are particularly useful for eliminating dysfunctions, increasing non-antigen-specific protection of an individual, eliminating stress-related effects, or otherwise (e.g. drug-induced immunosuppression or immunodeficiency and to have a regulatory effect between the immune, hormone and vascular systems.

The invention is further based on successful attenuation of a myxomavirus strain by passage over cell cultures, the virulent and / or immunizing properties of the myxomavirus strain being reduced or lost. Additional inactivation of the myxoma viruses can also take place by irradiation, exposure to heat or pH or, particularly preferably, by chemical treatment with beta-propiolactone. The monoparamity inducers are based on attenuated, lyophilized myxoma viruses, and individual virus components of a myxoma virus which are suitable for causing paramunizing activities in an organism are also included in the invention.

In the following, an embodiment of the manufacturing method according to the invention of the monoparamunity inducer based on an isolated and attenuated myxomavirus strain of the rabbit is to be presented via cell culture passage. The production process is not limited to this preferred strain, but is equally applicable to other rabbit myxomavirus strains. Also included in the present invention are recombinant forms of a myxoma virus strain which have been produced by means of genetic modification. Recombinant myxoma virus strains are preferred in which one or more sections in the genome which code for cytokine receptors have been modified by a modification in the form of an addition, substitution or deletion, the receptor properties of the cytokine receptor being lost as a result of the modification , These are preferably the gene segments which code for the receptors for interferons (1FN), interleukins (IL) and tumor necrosis factors (TFN), in particular for IFN-α-R, IFNγ-R, TNF-R, IL-1-R, IL - 2-R, IL-6-R and) L-12-R. Furthermore, the numerical values given here with regard to the incubation period or the number of passengers over cell cultures should not be interpreted as limiting. Minor modifications of these parameters and modifications which are obvious to the person skilled in the art and which also lead to the preparation of attenuated myxoma viruses are equally encompassed by this invention.

A preferred embodiment of the present invention relates to the successful attenuation of the myxomavirus strain M-2. The myxoma virus strain M-2 was isolated from European wild rabbits suffering from myxomatosis (Herrlich A., Mayr A. and Münz E .: "Die Pocken", 2nd edition, Georg Thieme Verlag, Stuttgart, 1967) The modified skin cells, which have been obtained from the subcutaneous tissue of the sick rabbit, are inoculated with chickens' eggs, preferably incubated for 10-12 days, after disruption to the chorioallantoic membrane (CAM). The myxoma viruses are inoculated over 2 to 6 passages, preferably over three passages. further increased or adapted on the chorioallantoic membrane (CAM passages; see Herrlich A., Mayr A. and Münz E .: "Die Pocken", 2nd edition, Georg Thieme Verlag, Stuttgart, 1967). The 2nd to 6th passage, preferably the third CAM passage, serves as the starting material for the further attenuation of the myxoma virus in cell cultures. Attenuation takes place after adaptation of the viruses in the chorioallantoic membrane (recognizable by typical foci on the chorioallantoic membrane) in 3 stages. In stage 1, 80 to 150, preferably 120, continuous so-called final dilution passages are run through in Vero cells (Nero cells, ATCC CCL-81). After going through these passages, the myxoma virus is weakened in its virulence.

In a second stage, after the 80th to 150th passage, preferably after the 120th passage in Vero cells, the virus suspension is transferred to so-called AVIVER cells and over 10 to 50 passages, preferably over 20 to 30, in particular over 25 Passages, continued. AVIVER cells are obtained by cell fusion between chicken embryo fibroblasts (FHE) and Vero cells and are called binary permanent cell culture.

The last passage via AVIVER cells, preferably the 25th passage, is transferred back to Vero cells and is continued in the 3rd stage of attenuation for a further 100 to 200 passages, preferably about 157 passages, in Vero cells. In this way, the myxoma virus is propagated over a total of more than 300 cell culture passages. After the 3rd stage of the multiplication of the myxomavirus in cell cultures, the myxomavirus is sufficiently attenuated. A fully synthetic medium is preferably used for culturing the VERO cell cultures and the AVIVER cells, the medium MEM (“minimal essential medium”) plus 5 to 20%, preferably 10% BMS (serum replacement medium) and 5 to 20, is particularly preferred. preferably 10% lactalbumin hydrolyzate After exchange with the growth medium, preferably MEM medium, 5 to 20%, preferably 10% lactal albumin hydrolyzate, without BMS or without fetal calf serum and without antibiotics is used as virus medium. All production processes are preferably at pH -Values from 7.0 to 8.0, preferably carried out at a pH of 7.25. Virus harvests with a titer of 10 ⁵⁰ to 10 ^7'5 , preferably of at least 10 ⁶ ' ⁵ TCID ₅₀ / ml (TCID ₅₀ = 50% tissue culture infectious dose), are preferably suitable as starting material for the preparation of the monoparamunity inducer PIND-MYXO according to the invention.

The multiplication of the myxoma virus leads to a typical cytopathic effect (cpE) in VERO cells, which is ultimately characterized by the destruction of the infected cells (lysis). With a seeding dose of approx. 10 MOI ("multiplicity of infection"), after a short period of detachment (1-2 days), reticulated cell structures occur for about 3 days and the cells are lysed after about 5 days. The 301st passage in VERO Cells had an infection titer of approximately 10 ^6.5 TCID ₅₀ / ml.

To inactivate the attenuated myxoma virus, chemical treatment with beta-propiolactone is carried out at a concentration of 0.01-1% beta-propiolactone, preferably at a concentration of 0.05% beta-propiolactone. This inactivation leads to a complete loss of any immunizing properties that may still be present, the parasite-specific activities not only being retained but actually increasing significantly.

For further processing of the attenuated and inactivated myxoma viruses into a myxoma virus monoparamunity inducer (PIND-MYXO), the virus starting material used for virus inactivation should have a virus titer of about 10 ⁵⁰ to 10 ⁷⁰ , preferably at least 10 ^{6 5} TCID ₅₀ / ml.

The cleaning is preferably carried out by centrifugation at a low speed (eg 1000 rpm). After centrifugation, 0.5-10% succinylated gelatin (for example polygeline, for example from Hausmann, St. Gallen / Switzerland), preferably 5% succinylated gelatin, is added. The resulting mixture can then be lyophilized in portions of 1.5 ml in appropriate sterile glass vials or ampoules and, if necessary, dissolved with distilled water (distilled water). A volume of 0.5-2 ml, preferably from 1.0 ml of aqua dest. dissolved lyophilisate corresponds to a vaccine dose for humans when administered intramuscularly (see also Mayr A. and Mayr. B .: "From Empiricism to Science", Veterinary Survey, Edition 57: 583-587, 2002).

Clinically, attenuation can be demonstrated by the loss of virulence for European domestic and wild rabbits (genus Orydolagus caniculus csp.), By a loss of contagiosity, by an almost complete narrowing of the host spectrum in cell cultures and by a change in the immunizing properties.

The lyophilized preparation can be stored at temperatures of preferably about + 4 ° C or at lower temperatures, preferably about -60 ° C, stable for an unlimited period.

Genetic testing of the prepared attenuated myxoma viruses has shown that multiple deletions had occurred in the myxoma virus genome. In the case of the parent strain M-2, the myxoma virus genome consists of a single linear deoxyribonucleic acid (DNA) with a total length of about 160 kilobases (kB), which codes for several hundred proteins (Herrlich A., Mayr A. and Münz E .: " Die Pocken ", 2nd ed., Georg Thieme Verlag, Stuttgart, 1967). The sequences of the terminally located" inverted repeats "(TIR) are approximately 11 kB of the genome section (McFadden, G and Graham, K .: "Modulation of cytokine networks by poxvirus", Virology, Ed. 5: 421-429, 1994).

It was found that the attenuation of the myxoma viruses via continuous Vero-cell passages led to a loss of the coding gene segments for the receptors

Interferon α and γ (IFN α, IFNγ), for the tumor necrosis factor (TNF) and for the interleukins

(IL-) 1, 2, 6 and 12 has come. Interestingly, these cytokines are parasitic defense factors of the non-specific immune system. The cytokines are neutralized by binding to the corresponding virus receptors, so that the virus can multiply uninhibitedly. The deletions of gene segments that code for the cytokine receptors mentioned above mainly affect the terminal regions of the DNA. In addition, it was also possible to detect deletions in the conserved part of the DNA that occurred during the AVIVER cell passages. These deletions affect two genes that code for an immune epitope and virulence gene. Such genetic modifications are presumably one of the reasons for the decrease in immunizing, i.e. antigen-specific activities and the simultaneous increase in paraspecific

Attenuated myxoma virus activities. The immunizing epitopes and the paraspecific or non-specific epitopes are competing. A decrease in the first-mentioned peptides or proteins therefore leads to an increase in the effect of the paraspecific activities. Residues of immunizing and virulence-increasing proteins are eliminated in the preparation of monoparamity inducers by the method described above for inactivating the attenuated myxoma viruses.

The monoparamunity inducer according to the invention, also called PIND-MYXO, is based on the use of attenuated myxoma viruses or their paramunizing components and, due to its paramunizing properties, is suitable for the following prophylactic or therapeutic indications in a patient:

- Infectious factor diseases and mixed infections, chronic manifestations of infectious processes, persistent recurrent infections and chemotherapy-resistant, bacterial and viral infections

- Defense weaknesses or dysregulations in the defense system of an organism

- neonatal threat of infection

- adjuvant therapy for certain tumor diseases, e.g. Prevention of metastasis, reduction of side effects through chemotherapy and radiation therapy

- Regulation of homeostasis between the hormone, circulatory, metabolic and nervous systems.

The paramunity inducers according to the invention can be administered to mammals, including humans, birds and reptiles, parenterally or locally. The local administration of paramunity inducers specifically stimulates the parasitic defense mechanisms in the mucous membranes and in the skin. In addition, there is also a certain systemic effect. Parenterally applied paramunizations, on the other hand, hardly influence the local defense mechanisms in the skin and mucous membrane, whereby they have a systemic effect.

Side effects did not occur even with numerous, continuously performed, parenteral applications in humans and animals. The following apply to the use of PIND-MYXO same indications in animals as in humans. At the same time ^', it is recommended in problem farms, especially in attitudes of horses to paramunisieren pigs, dogs and cats, the newborn immediately on the day of birth, and preferably on the first and possibly the second day after birth. The single dose is about 0.5 to 5 ml of the dissolved lyphilisate, in horses and pigs the single dose is preferably 2 ml and in dogs and cats preferably 0.5 ml with parenteral administration. According to the invention, it is advisable to administer PIND-MYXO one day before and / or at the same time as specific vaccinations parenterally to avoid side reactions and to support immunization when administering vaccines.

One embodiment of the invention relates to the production of a pharmaceutical composition for local application for the induction of paramunity in the skin and mucous membranes. The pharmaceutical composition preferably relates to a buccal or lozenge tablet based on components of an attenuated and inactivated Myxoma cell culture virus. The Buccal tablets according to the invention are preferably produced with the addition of sorbitol, polyethylene glycol 6.00, potassium hydrogen phosphate, tyrospirol tablet essence, Kollidon 25 and magnesium stearate. PIND-MYXO can also be administered nasally, rectally or vaginally with suitable carriers.

The following examples are preferred embodiments of the invention and serve to further explain the subject matter of the invention.

example 1

As a starting material for the production of the monoparamunity inducer PIND-MYXO according to the invention, the myxomavirus from the edematous subcutis of a European wild rabbit (genus Oryctolagus), which typically has myxomatosis, was isolated by incubation on the chorioallantoic membrane (CAM) for 10 days of incubated and broiled egg eggs three times according to the Herrlich et al. adapted to passages on the CAM (Herrlich A., Mayr A. and Münz E .: "Die Pocken", 2nd edition, Georg Thieme Verlag, Stuttgart, 1967). The third CAM passage was opened in a 1st stage VERO cells over 120 passages (ATCC CCL-81, WHO, American Type Culture Collection) adapted, in a second stage multiplied by 24 intermediate passages in AVIVER cell cultures and in the third phase cultivated in VERO cells. A total of about 300 Passages were carried out with the aim of attenuation. After these continuous final dilution passages, the originally virulent myxoma virus was attenuated. The attenuated myxoma virus is propagated in Vero cells. A fully synthetic medium consisting of MEM ("minimal essential medium") plus 10% BMS (serum replacement medium) and 10% lactalbumin hydrolyzate is used for the cultivation of the Vero cell cultures. After exchange with the cultivation medium, only MEM with 10% lactalbumin hydrolyzate is used without BMS or without fetal calf serum and without antibiotics. All production processes are carried out at pH values above 7.25. Virus harvests with titers above 10 ⁶⁵ TClD ₅₀ / ml serve as starting material for the production of the monoparamunity inducer PIND-MYXO according to the invention Inactivation of the virus harvest with 0.05% beta-propiolactone and coarse centrifugation, 5% succinylated gelatin (polygeline) is added to the virus material before lyophilization.

The lyophilized preparation keeps at room temperatures as well as at temperatures from approximately 4 ° C. to -80 ° C. and is preferably stable indefinitely at approximately 4 ° C. or also approximately -60 ° C. A volume of 1 ml of the in sterile aqua dest. dissolved lyophilisate corresponds to a vaccine dose. The application is deep intramuscular or local (see Examples 3, 4 and 5).

Example 2

In a manner analogous to that described in Example 1, the PIND-MYXO inducer according to the invention in dry form (lyophilisate not dissolved) is applied locally to the mucous membranes of the upper respiratory tract, preferably nasally, three times a day for prophylaxis or therapy (1 ml per application ) of multifactorial infections (e.g. flu infections).

Example 3

In an analogous manner, the PIND-MYXO inducer produced in Example 1 in liquid form is rubbed in according to the invention cutaneously for better blood circulation to the skin, for faster healing of wounds and for the treatment of varicose veins or chronic venous insufficiency (leg ulcers) in humans. For this purpose, the lyophilisate can be taken up, for example, in fat cream (eg Bepanthen, linola fat), whereby the pH should be slightly alkaline. This preparation should be freshly made for every application. The application is carried out several times a day by manually rubbing the undamaged skin. Open wounds can be treated by dripping the freshly loosened preparation onto the wound areas. Treatment should be done daily until healing. Example 4

Analogously, the monoparamunity inducer PIND-MYXO, prepared as in Example 1, is administered parenterally one day before and at the same time as a vaccination with classic, specific vaccines in order to avoid side reactions and to improve the vaccination success.

Example 5

Analogously, the monoparamunity inducer PIND-MYXO, produced as in Example 1, is processed into buccal or lozenges. The production and use of the lozenges for the local paramunization of the ear, nose and throat and oral mucosa is new and part of the invention. The activated oral mucous membranes not only cause a "homing" effect (migration of immune cells into mucous membranes of other organ systems), but also partial parenteral paramunization. The following manufacturing process has proven itself for the production of buccal or lozenges:

For the lyophilization, 5% Kollidon 25 (polyvinylpyrrolidone) is added to the liquid inducer material instead of gelatin. Urea, sorbitol, polyethylene glycol 6000 and magnesium stearate are required to produce the finished tablet. As a recipe for a tablet weighing 500.5 mg, the following is recommended:

PIND-MYXO lyophilisate 65 mg

Urea 50 mg

Sorbitol 267 mg

Polyethylene glycol 6000 118 mg

Magnesium stearate 0.5 mq

Tablet weight 500.5 mg

The patient should take 4-6 tablets per day at regular intervals to achieve optimal paramunization.

The following formulation of a pharmaceutical composition has proven itself for the production of Buccal tablets: PIND-MYXO lyophilisate 115 mg

Sorbitol 360 mg

Polyethylene glycol 6000 300 mg

Potassium dihydrogen phosphate (KH ₂ P0 ₄ ), anhydrous 2 mg

Disodium hydrogen phosphate (Na ₂ HP0), anhydrous 8 mg

Tyrospirol essence tablets 0.8 mg

Magnesium stearate 20 mq

Tablet weight 805.8 mg

The tablets slowly dissolve in the patient's mouth and can be swallowed after dissolution.

The clinical test results summarized in Tables 1 to 4 with the monoparamunity inducer according to the invention, based on the lyophilisate of the myxomavirus strain M-2, demonstrate the very good paramunizing activities of myxomavirus lyophiates in humans. These data are equally applicable to other mammals as well as birds and reptiles.

Table 1: Clinical results with a monoparamunity inducer from attenuated Myxoma cell culture virus in humans - prophylactic applications - (lyophilized inducer 1 OP (1 ml) intramuscularly)

Table 2: Clinical results with a monoparamunity inducer from attenuated Myxoma cell culture virus in humans - therapeutic use - (lyphilized inducer 1 OP (1 ml) intramuscularly)

Table 3: Effect of paramunization with myxoma inducer (PIND-MYXO) in patients with low immune parameters (7 days after PIND-Myxo application)

Table 4: Effect of paramunization with myxo-inducer (PIND-MYXO) in patients with increased immune parameters (7 days after PIND-MYXO application)

DISCLOSURES OF A DEPOSED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL (Rule 13 ^is PCT) The information below relates to the deposited microorganism or other biological material mentioned in the description on page 5, line 2ϊ ~ Z - B. LABELING OF THE DEPOSIT Further deposits are marked on an additional sheet name of the depository European Collection of Cell Cultures (ECACC)

Depository address (including zip code and country) Porton Down Salisbury SP4 OJG Wiltshire Great Britain Date of deposit Receipt number December 18, 2003 03121801 ADDITIONAL INFORMATION (if not applicable, please leave blank) Information will be continued on a separate sheet to all countries of destination for which such a declaration is possible, as far as this is legally possible according to the laws of the country of destination, a sample of the deposited microorganism is only given to an independent expert, in accordance with the respective patent legislation, e.g. EPO Rule 28 (4); UK Patent Rules 1995, Schedule 2, Parapraph 3, Australian Regulation 3.25 (3); Danish patent law sections 22 and 33 (3) and corresponding regulations of other destination states. COUNTRIES OF DESTINATION FOR WHICH INFORMATION IS PROVIDED (if the information does not apply to all countries of destination)

E. SUBMISSION OF INFORMATION (if not applicable, please leave blank. The information below will be submitted later to the International Office (please state the type of information, e.g. "receipt number of the deposit")

For use by the filing office only For use in the international office This sheet has been received by the international filing with the International Office D This sheet has been received on: ienste Jienste ⁰ te- Rossi

Plenipotentiary agent

Form PCT / RO / 134 (July 1998; reprinted January 2004) 25

Claims

1. Monoparamunity inducer based on paramunizing viruses or virus components, characterized in that the viruses or virus components come from an attenuated myxoma virus strain of the rabbit.

2. Monoparamunity inducer according to claim 1, characterized in that the attenuated myxomavirus strain has a modification in the form of an addition, substitution or deletion in one or more gene segments, coding for the formation of cytokine receptors, the modification having the receptor properties of the Cytokine receptor is lost.

3. Monoparamunity inducer according to claim 2, characterized in that the cytokine receptors are selected from the group of receptors for interferons (IFN), interleukins (IL) and tumor necrosis factor (TNF).

4. Monoparamunity inducer according to claim 3, characterized in that the cytokine receptors are selected from the group of receptors for IFNα-R, IFNγ-R, TNF-R, 1L-1-R, 1L-2-R, 1L-6 -R, IL-12-R.

5. Monoparamunity inducer according to one of claims 1 to 4, characterized in that the virus components comprise virus envelopes or aberrant forms of virus envelopes of an attenuated myxomavirus strain.

6. Monoparamunity inducer according to one of claims 1 to 5, characterized in that the myxomavirus strain is selected from the group consisting of M2, M7, Lausanne, Aust / Uriarra / Verg-86/1.

7. Monoparamunity inducer according to claim 6, characterized in that the myxomavirus strain is strain M-2 with the deposit number ECACC 03121801.

8. Monoparamunity inducer according to one of claims 1 to 7, characterized in that the myxomavirus strain has been attenuated in cell cultures.

9. Monoparamunity inducer according to claim 8, characterized in that the cell cultures are Vero monkey kidney cells and / or AVIVER cells.

10. Monoparamunity inducer according to one of claims 1 to 9, characterized in that the attenuated myxoma viruses have been inactivated.

11. Monoparamunity inducer according to claim 10, characterized in that the attenuated myxoma viruses have been inactivated with beta-propiolactone.

12. Monoparamunity inducer according to claim 11, characterized in that the concentration of beta-propiolactone is 0.01-1%.

13. Monoparamunity inducer according to claim 12, characterized in that the concentration of beta-propiolactone is 0.05%.

14. Monoparamunity inducer according to one of claims 1 to 13, characterized in that the myxoma viruses have been lyophilized.

15. Use of the Monoparamunitätsinducers according to any one of claims 1 to 14 for the manufacture of a pharmaceutical composition.

16. Use of the Monoparamunitätsinducers according to any one of claims 1 to 14 for the manufacture of a pharmaceutical composition for activating the paraspecific immune system in humans and animals.

17. Use of the Monoparamunitätsinducers according to any one of claims 1 to 14 for the manufacture of a pharmaceutical composition for parenteral treatment and / or for the prophylaxis of immune system dysfunctions, immunosuppression, immune deficiency disorders, dysfunctions of homeostasis between hormone, circulatory, metabolic and Nervous system, neonatal threat of infection, tumor diseases, viral diseases, bacterial diseases, therapy-resistant infectious factor diseases, viral and bacterial mixed infections, chronic manifestations of infectious processes, liver diseases of different origins, chronic skin diseases, herpes diseases, chronic hepatitis, flu infections, endotoxin.

18. Use according to any one of claims 15 to 17, wherein the treatment is carried out by local administration of the pharmaceutical composition over the skin or mucous membranes of a patient.

19. A pharmaceutical composition comprising a monoparamity inducer according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

20. Pharmaceutical composition according to claim 19, for local or parenteral application.

21. The pharmaceutical composition according to claim 19, wherein the composition is in the form of buccal and lozenges.

22. A method for producing a monoparamunity inducer based on an attenuated myxoma virus strain of the rabbit, comprising the steps of: isolating myxoma viruses from infected tissue of a rabbit typically suffering from generalized myxomatosis; Adaptation of the virus to a permissive cell system; Passage of the isolated viruses in a permissive cell culture until the virus is attenuated.

23. The method according to claim 22, wherein for adaptation the isolated viruses are vaccinated on the chorioallantoic membrane (CAM) of incubated chicken eggs.

24. The method according to claim 23, wherein the virus is propagated over 2 to 6 passages, preferably over 3 passages, on the CAM.

25. The method according to claim 22, wherein to isolate the myxoma viruses contained in the infected tissue, these are multiplied in a permissive cell system.

26. The method according to claim 25, characterized in that the virus is isolated by breeding on the chorioallantoic membrane (CAM) incubated chicken eggs.

27. The method according to claim 26, characterized in that the virus is subsequently adapted to the CAM over further passages, preferably over 2 further passages.

28. The method according to any one of claims 22 to 27, wherein the virus is passaged in a permanent cell culture.

29. The method according to claim 28, wherein the virus is passaged in a Vero cell culture.

30. The method according to claim 29, characterized in that the attenuation of the myxoma viruses takes place over 80 to 150 passages, preferably over 120 passages, in Vero cell cultures.

31. The method according to any one of claims 28 to 30, wherein the virus is passaged in a binary cell culture.

32. The method according to claim 31, wherein the virus is passaged in an AVIVER cell culture.

33. The method according to claim 32, characterized in that the passage takes place over 10 to 50 passages, preferably over 25 passages.

34. The method according to any one of claims 22 to 33, wherein the attenuated myxoma viruses are additionally propagated via further attenuation passages.

35. The method of claim 34, wherein the multiplication takes place in Vero monkey kidney cells.

36. The method according to claim 35, characterized in that the continuation takes place in Vero cells over 100 to 200 passages.

37. The method according to any one of claims 34 to 36, characterized in that the virus harvests of the cell culture have an infection titer of 10 ⁵ to 10 ^7.5 , preferably at least 10 ^{6 5} , TCID ₅₀ / ml.

38. The method according to any one of claims 22 to 37, wherein the attenuated myxoma viruses are additionally inactivated.

39. The method of claim 38, wherein the attenuated myxoma viruses are treated with beta-propiolactone for inactivation.

40. The method according to claim 39, characterized in that the concentration of beta-propiolactone is 0.01-1%.

41. The method according to claim 40, characterized in that the concentration of beta-propiolactone is 0.05%.

42. The method according to any one of claims 22 to 41, comprising the steps: isolating myxoma viruses from the infected tissue of the rabbit suffering from myxomatosis by multiplying chicken eggs incubated on the CAM and subsequently adapting the isolated myxoma virus to the CAM via a further 2 passages of attenuating the isolated Viruses by passage in Vero cell cultures, preferably over 120 passages, transfer of the viruses into the binary AVIVER cell culture with further attenuation of the virus, preferably over 24 intermediate passages, in this cell culture, subsequent retransfer of the virus to Vero monkey kidney cells, and multiplication of the attenuated Myxoma viruses through further attenuation passages in the Vero cells, preferably over about 150 passages.

43. Attenuiert.es Myxomavirus, obtainable by the method according to any one of claims 22 to 42.

44. Myxoma virus according to claim 43, deposited with the European Collection of Cell Cultures (ECACC) under the deposit number 03121801.

45. Myxomavirus according to claim 43 or 44, characterized in that the myxomavirus is genetically modified.

46. Myxomavirus according to claim 45, characterized in that the myxomavirus have a modification in the form of an addition, substitution or deletion in one or more gene segments coding for the formation of cytokine receptors, the receptor properties of the cytokine receptors being lost by the modification go.

47. Myxoma virus according to claim 46, characterized in that the cytokine receptors are selected from the group of receptors for interferons (IFN), interleukins (IL) and tumor necrosis factor (TNF).

48. Myxomavirus according to claim 47, characterized in that the cytokine receptors are selected from the group of receptors for IFNα-R, IFNγ-R, TNF-R, IL-1-R, IL-2-R, IL-6 -R, IL-12-R.